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低氧后处理诱导钙网蛋白表达上调的心肌保护机制

徐菲菲, 刘秀华^*, 张振英, 蔡莉蓉

中国人民解放军总医院病理生理研究室，北京 100853

摘要

钙网蛋白(calreticulin, CRT)是内质网(endoplasmic reticulum, ER)/ 肌浆网(sarcoplasmic reticulum, SR)中主要的Ca2+ 结合 分子伴侣，参与调节细胞Ca2+ 稳态和协助蛋白质折叠，在内源性保护现象——缺血后处理(ischemic postconditioning, I-postC) 过程中表达上调，但其发挥作用的分子机制尚未完全阐明。本工作在原代培养Sprague-Dawley (SD)乳鼠心肌细胞低氧/ 复 氧(hypoxia/reoxygenation, H/R)模型上，采用反义寡核苷酸(antisense oligodeoxynucleotides, AS-ODNs)抑制CRT 表达，观察 低氧后处理(hypoxic postconditioning, H-postC)过程中CRT 下游分子钙调神经磷酸酶(calcineurin, CaN)活性以及CaN、核转 录因子κB (nuclear factor kappa B, NFκB)、凋亡相关分子Bcl-2、Bax、C/EBP 同源蛋白(C/EBP homologous protein, CHOP) 等蛋白表达变化，及其与细胞保护的关系。心肌细胞随机分为6 组(n=4) ：对照组、低氧/ 复氧(H/R)组、低氧后处理(HpostC) 组、AS-ODNs 抑制CRT 表达(AS)组、AS + H/R 组和AS + H-postC 组，采用台盼蓝排斥实验、培养基乳酸脱氢酶 (lactate dehydrogenase, LDH)活性测定及流式细胞术检测细胞损伤；经Fluo-3/AM 染色，采用激光共聚焦显微镜测定细胞 浆游离Ca2+ 浓度；采用对硝基磷酸酚(p-nitrophenyl phosphate, PNPP)底物发色法测定CaN 活性；Western blot 检测相关蛋 白表达。结果显示：(1) H-postC 可减轻H/R 诱导的心肌细胞损伤，与H/R 组比较，细胞存活率升高17.1%，凋亡率和 LDH 漏出分别降低6.67% 和27.9% (P 均<0.05) ；(2) H-postC 轻度上调CRT，AS-ODNs 抑制CRT 表达后，部分消除后 处理的心肌保护作用，与H-postC 组相比，AS + H-postC 组细胞存活率降低8.98%、凋亡率和LDH 漏出分别升高1.74% 和13.6% (P 均<0.05)，而胞浆游离Ca2+ 浓度、CaN 活性、CaN 及NFκB 表达均未发生明显变化(P>0.05)，提示CRT 参与 后处理保护，但不是通过胞浆Ca2+-CaN 途径发挥作用；(3) H-postC 抑制促凋亡蛋白Bax、CHOP 表达(与H/R 组相比，分 别下调54% 和51%, P<0.05)，诱导抗凋亡蛋白Bcl-2 上调(与H/R 组相比，上调99%，P<0.05)，抑制CRT 表达部分减弱 H-postC 诱导的上述凋亡相关蛋白变化，提示H-postC 诱导CRT 上调可能通过调节凋亡相关蛋白表达参与心肌保护。综上 所述，H-postC 可降低胞浆Ca2+ 超载，减轻心肌细胞H/R 损伤；CRT 通过调节凋亡相关蛋白表达参与H-postC 心肌保护， 而不是通过Ca2+-CaN 途径发挥抗凋亡作用。

关键词： 钙网蛋白; 钙调神经磷酸酶; 钙; 离子超载; 缺血后处理; 低氧

分类号：R363.2

[Cardioprotective mechanism of calreticulin up-regulation induced by hypoxic postconditioning.] [Ariticle in Chinese]

XU Fei-Fei, LIU Xiu-Hua^*, ZHANG Zhen-Ying, CAI Li-Rong

Department of Pathophysiology, Chinese PLA General Hospital, Beijing, 100853, China

Abstract

Calreticulin (CRT) is an essential Ca2+-binding chaperone existing in endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR), and is involved in intracellular Ca2+ homeostasis and protein folding. Ischemic postconditioning (I-postC), a newly discovered endogenous protective phenomenon, induces CRT up-regulation. The present study aimed to investigate the cardioprotective mechanism of CRT up-regulation induced by hypoxic postconditioning (H-postC). Primary cultured neonatal rat cardiomyocytes were exposed to 2 h of hypoxia followed by 24 h of reoxygenation. Postconditioning was carried out by two cycles of 10 min of reoxygenation and 20 min of rehypoxia after 2 h of hypoxia. Antisense oligodeoxynucleotides (AS-ODNs) were used to inhibit CRT expression 36 h before hypoxia. Cardiomyocytes were randomly divided into 6 groups as follows (n=4): control, hypoxia/reoxygenation (H/R), HpostC, AS, AS + H/R, and AS + H-postC. Morphological studies, lactate dehydrogenase (LDH) activity assay in culture medium, and flow cytometry were used to detect cardiomyocyte necrosis and apoptosis. Intracellular Ca2+ concentration was detected by fluorescent Fluo-3/AM staining through laser confocal microscope, and p-nitrophenyl phosphate (PNPP) was used as substrate to measure calcineurin (CaN) activity. The expression of CRT, CaN, nuclear factor kappa B (NFκB) and apoptosis-related proteins, such as Bcl-2, Bax and C/EBP homologous protein (CHOP) were detected by Western blot. The results were as follows. (1) H-postC protected neonatal cardiomyocytes from H/R injury. Compared with H/R group, cell survival rate increased by 17.1%, apoptotic rate and LDH leakage decreased by 6.67% and 27.9% in H-postC group, respectively (P<0.05). (2) H-postC induced mild up-regulation of CRT expression. Inhibition of CRT by AS-ODNs attenuated the cardioprotection of H-postC partly. Compared with H-postC group, cell survival rate decreased by 8.98%, and apoptotic rate and LDH leakage increased by 1.74% and 13.6% in AS + H-postC group, respectively (P<0.05), but intracellular Ca2+ concentration, CaN activity, and expression of CaN and NFκB did not change significantly (P>0.05), suggesting that CRT participates in endogenous protection, not through Ca2+-CaN pathway. (3) H-postC inhibited the expression of pro-apoptosis proteins such as Bax and CHOP, but induced up-regulation of anti-apoptosis protein Bcl-2. Inhibition of CRT by AS-ODNs partly inhibited the changes in apoptosis-related proteins expression induced by H-postC, suggesting that CRT participates in the anti-apoptosis effect of H-postC through regulating expression of apoptosis-related proteins. These results indicate that CRT up-regulation induced by H-postC is involved in the cardioprotection through regulating expression of apoptosis-related proteins, not through Ca2+-CaN pathway in neonatal cardiomyocytes.

Key words: calreticulin; calcineurin; calcium; iron overload; ischemic postconditioning; hypoxia

收稿日期：2008-07-18　　录用日期：2008-11-20

通讯作者：刘秀华　　E-mail: xiuhualiu98@yahoo.com.cn

引用本文：

徐菲菲, 刘秀华, 张振英, 蔡莉蓉. 低氧后处理诱导钙网蛋白表达上调的心肌保护机制[J]. 生理学报 2009; 61 (1): 35-42.

XU Fei-Fei, LIU Xiu-Hua, ZHANG Zhen-Ying, CAI Li-Rong. [Cardioprotective mechanism of calreticulin up-regulation induced by hypoxic postconditioning.] [Ariticle in Chinese] . Acta Physiol Sin 2009; 61 (1): 35-42 (in Chinese with English abstract).